Nucleic Acids Research Advance Access originally published online on December 20, 2006
Nucleic Acids Research 2007 35(4):e21; doi:10.1093/nar/gkl917
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Nucleic Acids Research, 2007, Vol. 35, No. 4 e21
© 2006 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Methods Online |
Use of signal sequences as an in situ removable sequence element to stimulate protein synthesis in cell-free extracts
1Interdisciplinary Program for Biochemical Engineering and Biotechnology, College of Engineering, Seoul National University, Seoul 151-742, Korea, 2School of Chemical and Biological Engineering, College of Engineering, Seoul National University, Seoul 151-742, Korea and 3Department of Fine Chemical Engineering and Chemistry, Chungnam National University, Daejeon 305-764, Korea
*To whom correspondence should be addressed. Tel: +82 42 821 5899; Fax: +82 42 823 7692; Email: dmkim{at}cnu.ac.kr
Received September 14, 2006. Revised October 3, 2006. Accepted October 17, 2006.
This study developed a method to boost the expression of recombinant proteins in a cell-free protein synthesis system without leaving additional amino acid residues. It was found that the nucleotide sequences of the signal peptides serve as an efficient downstream box to stimulate protein synthesis when they were fused upstream of the target genes. The extent of stimulation was critically affected by the identity of the second codons of the signal sequences. Moreover, the yield of the synthesized protein was enhanced by as much as 10 times in the presence of an optimal second codon. The signal peptides were in situ cleaved and the target proteins were produced in their native sizes by carrying out the cell-free synthesis reactions in the presence of Triton X-100, most likely through the activation of signal peptidase in the S30 extract. The amplification of the template DNA and the addition of the signal sequences were accomplished by PCR. Hence, elevated levels of recombinant proteins were generated within several hours.